The origin and development of human tumors begins at the molecular level and involves a complex multi-step process. A common feature of many tumor cells is the ability to progress through the cell division cycle under conditions where normal cells would be quiescent or proliferating at a reduced rate. Thus, the molecular pathways controlling the cell division cycle must inevitably interact with pathways regulating cell growth, and are a likely target of oncogenic events. In previous studies, the P.I. has discovered that in normal human fibroblasts, the primary cell division cycle regulators, cyclin-dependent kinases (CDKs), exist predominantly in multiple quaternary complexes -- each composed of a CDK, cyclin, proliferating cell nuclear antigen (PCNA) and p21. Transformed cells show striking changes in the subunit composition of CDKs including the loss of PCNA and p21. Recently, we and others have shown that p21 encodes an universal inhibitor of CDKs, and that p21's expression is regulated at least in part by the tumor suppressor p53. With a long term goal of understanding the mechanism underlying cell cycle control and tumor suppression, we propose to determine the function of the newly discovered p53-regulated CDK inhibitor p21. We will perform following experiments to study the function of p21 in cell cycle control at the protein level: (a) generation of p21 specific antibodies, (b) expression and function of p21 protein during the cell cycle, (c) analysis of posttranscriptional regulation of p21, and (d) identification of cellular protein(s) that interact with p21. Tumor suppressors p53 and pRb both act as negative regulators of cell growth and cell cycle. We suggest that p21 not only mediates p53-, but also pRb-induced cell growth arrest. The specific questions to be addressed in this aim are; (a) Does inactivation of p21 function block p53-induced cell cycle arrest? (b) Does cyclin or CDK gene amplification and overexpression titrate p21 and override p53- and p21-induced cell growth arrest? And, (c) is the overriding of pRb and p107-induced cell growth arrest by cyclin overexpression mediated by titration of p21? The identification of p21 as a major transcript induced by wild type p53 function suggests the possibility that p21 is the major mediator of p53 tumor suppression function and that loss of both functional copies of p21 would have the same consequence as the loss of both copies of p53. To better understand the regulation of p21 gene expression by p53 an to directly test potential tumor suppression function of p21, we plan to further characterize p21's expression and search for p21 gene mutations in a variety of human cancer cell lines and tumors. The objectives of this aim are; (a) Northern analysis of p21 gene expression in a variety of cancer and tumor cells, (b) PCR amplification and sequencing analysis of p21, and (c) functional assays of identified p21 mutations.